Hücre Kültürlerindeki Mycoplsma Kontaminasyonlarının Fluorescense (DAPI) ve Aceto-Orcein Boyama Yöntemleri ile Tespiti Çalışmaları

Öz

Hücre kültürlerinin mikoplazma kontaminasyonlarının tespiti için kullanılan aceto-orcein ve floresan DAPI ile boyama yönteminin bazı özellikleri karşılaştırmalı olarak incelendi: 1- Laboratuvarımız stoklarında mevcut hücre kültürleri bu iki metot ile test edildi; 2- Birinci aşamada mikoplazma suşu ile kontamine edildi ve gene sözkonusu iki metot ile test edildiler; 3-Mikoplazmadan ari Vero, FL ve BHK21 An30 hücre kültürleri, kontamine olduğu birinci aşamada tespit edilmiş olan BHK21 An14 hücre kültürünün vasatı ile ayrı ayrı 1/10 ve 1/100 dilusyonlarda enfekte edildi ve iki metotla test edildiler. Sonuçta floresan DAPI boyama yönteminin aceto-orcein boyama yöntemine nisbetle daha duyarlı olduğu tespit edildi. Ancak, literatürde bildirilen bazı hatalı pozitif ve negatif reaksiyonların varlığı gözönüne alınarak, daha başarılı sonuç almak için iki yöntemin beraberce kullanılması tavsiye edildi.

Anahtar Kelimeler

• Hücre Kültürü
• Hücre Kültüründe Mycoplsma Kontaminasyonu
• Mycoplsma Kontaminasyonu
• Floresan DAPI Boyama Yöntemi
• Aceto-Orcein Boyama Yöntemi
• Mikoplazma Kontaminasyonları
• Fluorescense (DAPI) Boyama Yöntemi
• 1990
• S. İsmet GÜRHAN
• Aysun ÖZTÜRKMEN
• Etlik Veteriner Mİkrobiyoloji Dergisi

A study on the detectlon of mycoplasma contaminations in cell cultures by using fluorescent DAPI and aceto-orcein staining techniques

Öz

Some aspects of the detection of mycoplasma contaminations in ·cell culturs both by fluorescent DAPI stain and aceto-orcein stain have been studied comperatively : 1 - The cell cultures already present in our laboratory's stocks have been tested by those two methods. 2 - Three of mycoplasma-free cell cultures tested in the first step have been artificially infected with three different mycoplasma strains arid stained by two techniques comperatively; 3_ Mycoplasma-free Vero, FL and BHK21 An30 cell cultures. have been infected with the medium of mycoplasma contaminated BHK21,An14 cell culture in two different dilutions (1 /10 and 1 /100) and after 24 hours of incubation stained with two methods separetely. As a result, fluorescent DAPI staining assay has been estimated more sensitie than aceto-orcein staining. However by taking into concideration some false positive and negative reactions mentioned in the literature, the combination of two techniques has been advised for more satisfactory detection.

Kaynakça

1. CHEN, T.R,, (1977) ·: In situ Detection of Mycoplasma Contamination in Cell Cultures By Fluorecent Hoechst 33258 Stoin. Exp. Cell Res., 104, 25'5-262.
2. FOGH. J. and FOGH, .H., (1965) : A Method for Direct Demonstration of PPLO in Cultured Cells. Proc. Soc. Exp. Biol. N.Y. 117, 899-901 .
3. Food and Drug Administration, Bethesda MD 20205 Nov. 18: ·1987, «.Points to Cohsider in the Characterization of Cell-Lines· Used to Produce Biologicals. ».
4. McGARRITY, G.J., PHILLIPS, D. and VAIDYA, A., (1980) : Detection of M. hyorhinis infection .in Cell Respiratory Gultures. Cytogenet Cell Genet., 27, 194-196. McGARRITY, GJ. STEINER. T. and VANAMAN. V. (1983) : «Use of lndi cator Geli ·Lines ,for Recovery and ldentification of · cell Culture Mycoplasmas; Methods in Myccoplasmology Vol. 2, pp. 167-190; Acad. Press. Inc. N.Y .• London.
5. MOWLES; .J.M: (1989) .: The Use of Ciprofloxacin for the Elimination of Mycoplasma from Naturally İnfected Cell-Lines. Accepted for Publication : in Cytotechnology,
6. POLAK, A.A, ( 1983) : Detection and Elimination of Mycoplasmas in Cell Cultures. Thesis, RIVM; Nederlands.
7. PRECIOUS, B. and R·USSELL, W.C. (1987) ' Growth, Purification and Titrat lion of -Adenoviruses. Virology, a practical approaoh ed. B.W.J. Mahy. IRL Press Ltd. Oxford,Washington D.G.
8. ROBINSON, L.B .. WICHELHAUSEN, R:B. and RAZINAN, B., (1956) : Cantamination of Human Cell Cultures by Fleuro-pneumonia Like Organisms. 124, 1147-1150.

9. RUSSEL. W.C .. NEWMAN, C. and WILLIAMSON, D.H, (1975) : A Simp'e Cytochemical Technique for Demonstration of DNA . in Cel's lnfected with Mycoplasmas and Viruses. Nature (London) 253, 431-462.
10. STRANBRIDGE, E. and SCHNEIDER, E. L. : «The Need for Non Culturcl Methods for the Detection on Mycoplasma Contaminan:s» Joint WHO/IABS Symposium on the Standardization of Cell Substrates for the Production of Virus Vaccines. Geneva. Dec. 1976. Develop. Biol. Standard. Vol. 37, pp. 191- 200. (S. Karger, Basel. 1977).
11. World Health Organization Technical Report Series 747, WHO Geneva, 1987. (Acceptability of Cell Substrates for Production of Biologicals>